Herpes Simplex virus as a vector

ABSTRACT

A foreign gene is inserted into a viral genome under the control of promoter-regulatory regions of the genome, thus providing a vector for the expression of the foreign gene. DNA constructs, plasmid vectors containing the constructs useful for expression of the foreign gene, recombinant viruses produced with the vector, and associated methods are disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation of copending, commonly assigned application Ser.No. 07/455,771 filed Dec. 28, 1989, now abandoned, which is acontinuation of application Ser. No. 06/616,930 filed Jun. 4, 1984, nowabandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to expression vectors and, more particularly,this invention relates to the use of a viral genome as a vector forexpression of foreign genes.

2. Brief Description of the Prior Art

Infection of susceptible cells with certain viruses, such as the HerpesSimplex virus (HSV), for example, typically results in shut-off of hostprotein synthesis. The shut-off occurs in two stages. The initial stageis very likely caused by a structural protein of the virus, and geneticstudies indicate that this activity is not essential for virus growth. Asecond, irreversible inhibition occurs during the viral reproductivecycle as a consequence of expression of viral gene products. Availabledata based on chemical enucleation with actinomycin D or physicalenucleation with the aid of cytocholasin B suggests that the inhibitionis at least in part at the translational level.

Although Herpes Simplex virus, type 1 (HSV-1) was previously known toinduce some host genes, and particularly foreign genes such asovalbumin, for example, placed under control of viral regulatory regionsand introduced into cells by transfection, their expression is selectiveand transient. It was previously believed that the inhibitory machineryof wild type virus would not permit sustained expression of a foreigngene introduced into the viral genome.

SUMMARY OF THE INVENTION

According to the present invention, a foreign gene is inserted into aviral genome under the control of promoter-regulatory regions of thegenome; the viral genome thus becomes a vector for expression of theforeign gene in infected cells. Such expression is regulated by thepromoter-regulatory regions of the genome.

Thus, a viral genome is genetically engineered to render it useful forserial propagation of the gene along with the viral genome and for thesustained expression of foreign genes in a suitable host notwithstandingthe shut-off of protein synthesis directed by host chromosomes.

The invention is exemplified by the use of HSV-1 as a vector for theexpression of hepatitis B surface antigen (HBsAg). In order to enableand to regulate its expression, the HBsAg gene is placed under thecontrol of HSV gene promoter-regulatory regions. This construct is theninserted into the thymidine kinase (TK) gene of the viral genome.Additionally, a deletion may be made in the TK gene in order toinactivate it. The resulting DNA construct is cotransfected with theintact DNA of a suitable HSV-1 strain, and TK- progeny are selected.Such progeny are found to contain recombinants containing both thedeletion and the HBsAg insert. Cultures of such recombinants are foundto effectively produce HBsAg over sustained time periods.

According to the invention, a DNA construct, a viral vector containingthe DNA construct useful for expression of foreign genes, a method ofpreparing such a construct and vector, a method of expression using thevector, and specific plasmids and recombinant viruses are provided.

This invention makes possible simultaneous introduction and synchronousexpression of foreign genes in large scale cell cultures without priorselection of uniquely transformed genes. It makes possible theexpression of genes which normally are poorly expressed, and genes oforganisms that are either hazardous or cannot be cultivated in culture.

Other objects and advantages of the invention will be apparent to thoseskilled in the art from the following detailed description, taken inconjunction with the Figures and the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a flow sheet schematically illustrating the construction of arecombinant Herpes Simplex virus containing a β-TK promoter-regulatedhepatitis B surface antigen gene;

FIG. 2 is a graphical representation demonstrating the buoyant densityin cesium chloride gradient of purified expressed HBsAg in cellsinfected with the virus of FIG. 1;

FIG. 3 is an electron micrograph of purified expressed HBsAg obtainedfrom cells infected with the virus of FIG. 1;

FIG. 4 is a flow sheet schematically illustrating the construction of arecombinant plasmid containing chimeric αICP4-HBsAg gene; and,

FIG. 5 is a flow sheet depicting cotransfection of the plasmid of FIG. 4with intact viral DNA and selection of a recombinant Herpes Simplexvirus containing αICP4-HBsAg gene.

DETAILED DESCRIPTION OF THE INVENTION General Statement of the Invention

To convert a Herpes Simplex virus (HSV)to a vector according to theinvention, it is sufficient to recombine into its DNA a foreign genelinked to a suitable viral promoter. The foreign gene must have its owntranscription termination-poly-adenylation signal, or a new such signalmust be provided.

The foreign gene should contain a complete coding sequence. If theforeign gene terminates beyond the transcription termination signal andif downstream from the transcription termination signal there existsanother promoter, then the structural sequences of the TK gene of theHSV must be modified so that the TK gene cannot be expressed from thatpromoter.

In order to recombine the foreign gene into the virus, it is necessaryto have homologous flanking sequences through which the gene wouldrecombine at the desired location and a system for selecting the desiredrecombinant. In this instance the flanking sequences consist of portionsof the domain of the viral TK gene and, because selection for aninactive TK gene is being effected, any nucleotide analog (e.g. a drug)that is uniquely or mainly phosphorylated by the TK gene can be used forselection of the recombinant, TK- virus.

In the case of HSV, the TK gene is a highly desirable location forinsertion of the foreign gene because it allows selection ofrecombinants occurring at very low frequencies. Also, the position ofthe TK gene within the HSV genome can be changed (by known methods)prior to insertion of the foreign gene.

However, it is not necessary that the insertion of the foreign gene bemade in the TK gene; insertion may be made at any available non-lethalsite and selected by the use of another selectable marker in the HSVgenome.

HSV genes form three major groups designated as α, β and γ, theexpression of which is coordinately regulated and sequentially orderedin a cascade fashion. It is known that for most α and some β genes thepromoter and regulatory domains are located upstream from the site ofinitiation of transcription. Specifically, chimeric genes constructed byfusion of promoter-regulatory domains of α gene (e.g., the genespecifying infected cell protein (ICP) Nos. 0, 4, 27 or 22) to the 5'transcribed non-coding and coding sequences of other genes are regulatedas α or β genes, respectively.

Therefore, according to the invention, a DNA construct is preparedwherein a foreign gene containing its complete structural sequence,flanked on one end with a promoter-regulatory region of a viral gene andon the other with a suitable transcription termination signal, ispermanently integrated into a HSV genome.

Such a construct will have the ability to perpetuate the foreign gene inthe viral genome, and to express the foreign gene in cells infected withthe virus carrying the recombined viral genome.

Exemplary Embodiments of the Invention Genetic Engineering of HSV-1Vectors Carrying α- and β-Regulated HBsAg

In exemplary embodiments of the invention, the structural sequence and25 base pairs of the 5' transcribed non-coding region of the genespecifying HBsAg is placed under the control of the α promoter of ICP4or the β promoter of the viral thymidine kinase (TK) genes of an HSV-1genome by fusion of the 5' transcribed non-coding region and codingregion of the HBsAg gene to the respective promoter-regulatory regionsof the HSV-1 genes. The chimeric constructs are then inserted into the5' transcribed non-coding region of the TK gene by homologousrecombination through flanking sequences. Cells infected withrecombinants carrying the chimeric genes are found to produce andexcrete HBsAg into extra-cellular medium for at least 12 hours.

The temporal patterns of expression and the observation that HBsAglinked to the α-promoter-regulatory region was regulated as an α geneindicate that HBsAg gene chimeras inserted into the virus are regulatedas viral genes.

The nucleotide sequence of the TK gene of HSV-1 has been published. SeeWagner et al, Proc. Natl. Acad. Sci. USA, vol. 78, pp. 1441-1445 (1981)and McKnight, Nucleic Acids Res., Vol. 8, pp. 5949-5964 (1980). Thesingle Bgl II site within the domain of the TK gene is located withinthe 5' transcribed but not translated region of the TK gene. Deletion atthis site does not affect the promoter function of the region locatedupstream from that site.

Suitable procedures useful for the insertion of a foreign gene into anHSV-1 genome and the deletion of a portion of the HSV-1 TK geneaccording to the invention are described by Mocarski et al, Cell, Vol.22, pp. 243-255 (1980); Post et al, Proc. Natl. Acad. Sci. USA, Vol. 77,No. 7, pp. 4201-4205 (1980); Post et al, Cell, Vol. 24, pp. 555-565(1981); and Post et al, Cell, Vol. 25, pp. 227-232 (1981), as well as inEuropean Patent Publication No. 74,808 (Mar. 23, 1983) and Roizman, etal. U.S. Pat. No. 4,769,331 (Sep. 6, 1988). The disclosures of theforegoing publications are hereby incorporated by reference.

Specifically, both the β TK and the α ICP4 regulated HBsAg are insertedinto the Bgl II site of the TK gene interrupting the 5' transcribednon-coding region of that gene. The chimeric fragments are thencotransfected with intact HSV DNA, and TK- recombinants carrying theHBsAg gene produced by homologous recombination through flankingsequences are selected by plating on tk- cells in the presence of BuDR,which inactivates the TK+ viral progeny.

Because the DNA fragment carrying the HBsAg gene appears to contain atits terminus 3' to the gene a promoter which substitutes for the TKpromoter and maintains the TK+ phenotype, it is necessary to inactivatethe TK gene in the chimeric construct by a small deletion at the Sac Isite within the coding sequences of that gene.

This procedure allows the construction of HSV containing β-promoterregulated HBsAg, and is described in more detail below.

Further, to differentiate the expression of α-regulated HBsAg from thatof β-recombinant viruses, a construction involving ligation of thepromoter-regulatory region of the α4 gene of HSV-1 to a DNA fragmentcontaining the HBsAg gene was made. The chimeric gene was cloned intothe Bgl II site of the TK gene, and recombined with intact viral DNA bycotransfection to produce a recombinant virus. The details and resultsare set forth in detail below.

With reference now to FIGS. 1-5, the construction of two recombinantHerpes Simplex viruses containing an inserted coding sequence forhepatitis B surface antigen (HBsAg), and the expression of this proteinfrom the virus, are described in detail.

Construction and Expression of an HSV-1 Recombinant Containing aChimeric β-TK Promoter Regulated HBsAg

Plasmid pRB 3222 was made from plasmid pRB 103 (deposited on Jun. 4,1984 as ATCC Accession No. 39718) carrying the Bam HI Q fragment ofHSV-1 strain F[HSV-1(F)] (ATCC Accession No. VR733) by Bal 31exonuclease digestion to remove approximately 200 base pairs at theunique Sac I site in order to inactivate the TK gene. In thisconstruction, the initiation codon for HBsAg is located 25 base pairsdownstream from the Xho I site and close to the promoter region of TKgene. Therefore, the β-regulated gene was about 80 base pairs downstreamfrom the transcription-initiation site derived from the β TK gene,whereas the initiation codon for HBsAg in the α-regulated gene (below),was about 60 base pairs from the transcription initiation codon derivedfrom the αICP4 gene.

The DNA of plasmid pCP 10 (deposited on Jun. 4, 1984 as ATCC AccessionNo. 39717) was cleaved with Xho I and Bgl II and the resulting digestwas subjected to electrophoresis in 5% polyacrylamide gel. The Xho I-BglII fragment (approximately 1.7 kb, which contained the coding sequencefor HBsAg) was then purified from the polyacrylamide gel and the terminiof DNA fragments were filled with T4 DNA polymerase, ligated to Bgl IIlinkers, cleaved with Bgl II to produce cohesive ends, and cloned intothe Bgl II site of pRB 3222.

The resulting plasmid containing the HBsAg gene in the correcttranscriptional orientation relative to the TK promoter-regulatoryregion (as determined from a Bam HI DNA restriction pattern) isdesignated herein as pRB 3223 (deposited on Jun. 4, 1984 as ATCCAccession No. 39716).

Recombinant plasmid pRB 3223 was linearized with Pvu II, and 0.1-0.5 μgof the plasmid was then cotransfected with 0.5 μg HSV-1 (F) DNA intorabbit skin cells and plaque purifications of resulting recombinantviruses were carried out as described by Ruyechan et al, J. Virol, Vol.29, pp. 677-697 (1979). The resulting TK-recombinant viruses were thenselected on 143 tk- cell line in the presence of a medium containingmixture 199 lacking thymine but supplemented with 3% calf serum and 40μg/ml BuDR (bromouracil deoxyriboside).

As predicted, the resulting TK- progeny contained a recombinant whichrecombined only the deletion in the Sac I site (designated R3222) andanother that recombined both the deletion and the HBsAg gene insertion.The latter recombinant is designated R3223 (deposited on Jun. 4, 1984asATCC Accession No. VR2086).

Recombinant virus R3223 was differentiated from R3222 by digestion ofthe recombinant viral DNAs with Eco RI restriction endonuclease. Therespective DNA organizations of these viruses were further confirmed bySouthern blot analysis.

Table 1, below, and FIG. 2 demonstrate expression and excretion of HBsAgby recombinant virus R3223. Vero cells in 25 cm² flasks were exposed toR3223 (2 pfu/cell) for one hour and then incubated at 37° C. in a mediumconsisting of maintenance mixture 199 supplemented with 1% calf serum.At the times indicated in Table 1, 5 ml of the maintenance medium in oneflask were removed, the cells were washed three times with 5 ml each ofphosphate buffered saline (PBS) (0.15 M NaCl, 8.2 mM Na₂ HPO₄, 1.5 mMKH₂ PO₄, and 2.5 mM KCl), harvested in 1 ml of PBS, frozen-thawed threetimes and centrifuged in an Eppendorf microcentrifuge.

The supernatant fluid containing the cell lysate was then brought to avolume of 5 ml with PBS. Portions containing 200 μl of medium andinfected cells were then assayed for the presence of HBsAg with theAbbott Laboratories ELISA diagnostic kit sold under the trademarkAUSZYME II, according to the procedure recommended by the manufacturer,as follows.

Extracellular medium and lysates from recombinant and parent virus[HSV-1(F)] infected cells were mixed with beads coated with guinea pigantibody to HBsAg. After incubation, the beads were reacted withperoxidase-conjugated antibody to HBsAg. The presence of HBsAg wasmeasured spectrophotometrically at 492 nm.

As seen in Table 1, as early as four hours post-infection, HBsAg wasdetected in infected cell lysate but not in the medium. At eight hourspostinfection, HBsAg was detected both in lysates of infected cells andin the extracellular medium. At twelve hours postinfection, the bulk ofHBsAg was recovered from the extracellular medium. However, the amountsof HBsAg in infected cells were about the same as those detected atearlier times postinfection.

The amounts of HBsAg accumulating in the infected cells reached peaklevels approximately 15 fold higher than the background levels obtainedwith medium and lysates of cells infected with HSV-1(F) at or before 8hr postinfection and did not significantly increase thereafter. Theamounts of HBsAg detected in the extracellular medium increased withtime indicating that HBsAg was excreted and accumulated outside theinfected cell. The patterns of accumulation of α- and β- regulated HBsAgwere similar and in accord with the observation that the kinetics ofaccumulation of α- and β-regulated TK genes expressed by virusescarrying these genes were also similar.

HBsAg was not detected in wild type [HSV-1(F)] virus infected cells orextracellular medium. The observation that HBsAg is excreted was alsoshown in cells infected with vaccinia virus carrying the HBsAg gene.

                  TABLE 1                                                         ______________________________________                                        Expression of α- and β-regulated HBsAg                             in Cells Infected with Recombinant and Parent Viruses                         Hrs   HSV-1(F)     R3223        R3225                                         post           Cell           Cell         Cell                               Infect.                                                                             Medium   lysate  Medium lysate                                                                              Medium lysate                             ______________________________________                                        4      0.08*   0.07    0.42   0.57  0.58   0.56                               8     0.08     0.07    3.69   0.95  3.31   1.06                                12   0.09     0.07    6.33   1.26  5.06   1.20                               ______________________________________                                         *Optical density units.                                                  

FIG. 2 graphically represents the results of a study carried out tocharacterize excreted HBsAg obtained as described above. Vero cells wereinfected with R3223 at 2 pfu/cell. At 12 hours postinfection, 9 ml ofmaintenance medium was harvested and centrifuged at 36,000 rpm for 20hours at 4° C. as Beckman SW41 rotor. The resulting pellet was suspendedin 0.5 ml of the same medium and then 200 μl were layered on top of a1.1-1.5 g/ml isopycnic CsCl density gradient and centrifuged at 36,000rpm for 36 hours at 25° C. in a Beckman SW41 rotor. Fractions (0.5 ml)were collected from the bottom of the centrifuge tube and diluted with1:10 with PBS. The fractions were assayed for the presence of HBsAg asdetailed above with reference to Table 1.

As seen in FIG. 2, the HBsAg banded at a density of 1.17 g/ml. It isknown that in serum, HBsAg forms spherical particles with diametersbetween 18 and 25 nm (Dane et al, Lancet, Vol. 1, p. 695, 1970). As seenin FIG. 3, particles similar in dimension (i.e. 18-25 nm) to Daneparticles were detected by electron microscope examination of the peakfraction (which was negatively stained with 2% phosphotungstic acid),thus confirming the character of the excreted HBsAg.

The foregoing description details the preparation of a recombinant HSV-1viral plasmid carrying the coding sequence for HBsAg, and the effectiveuse of the plasmid as a vector for expressing that protein. In theconstruction of FIGS. 1-3, the HBsAg fragment was placed under thecontrol of a β promoter region of the virus.

Construction and Expression of an HSV-1 Recombinant Containing anαICP4-HBsAg Gene

The following describes the construction of a recombinant HSV-1 plasmidand a recombinant virus carrying the HBsAg coding sequence and gene,respectively, under the control of an α promoter.

FIG. 4 demonstrates the construction of a recombinant Herpes Simplexvirus containing chimeric αICP4-HbsAg gene. The construction involvedligation of the promoter-regulatory region of the α4 gene of HSV-1 tothe DNA fragment containing the HBsAg gene described above.Specifically, the Bam HI Q fragment containing the HSV-1 (F) TK genefrom pRB 103 was inserted into the Xho I site of plasmid pRB 14, whichwas constructed from pBR 322 (ATCC Accession No. 31344) by replacementof the Eco RI-Pvu II fragment with Xho I linker obtained from NewEngland Biolabs, Cambridge, Mass. USA.

The resulting recombined plasmid is designated herein as pRB 3148.Plasmid pRB 3159 was then constructed by cloning the Hind III-Hae IIIfragment (containing the polylinker) from pUC 13 into the unique Bgl IIsite of the HSV-1 (F) Bam HI Q such that the Bam HI site was closest tothe structural sequence of the TK gene, whereas the Sal I site wasclosest to the transcription initiation site of that gene. Plasmid pUC13 was selected as a suitable source for the fragment containing thepolylinker, but suitable polylinkers are commercially available.

Plasmid pRB 3166 was constructed from pRB 3159 by digesting with Sac Iand religating to delete the Sac I fragment containing the Bgl II-Sac Ifragment of Bam HI Q.

In the last step, two fragments were cloned into pRB 3166 to yieldplasmid pRB 3225. First, the Bam HI-Pvu II fragment from pRB 403(deposited on Jun. 4, 1984 as ATCC Accession No. 39719) containing thepromoter-regulatory domain of the αICP4 gene was cloned into the Sal Isite of the polylinker sequence such that the transcription initiationsite of αICP4 in the Bam HI-Pvu II fragment was close to the Xba I site.Lastly, the Bgl II fragment containing the HBsAg gene from pRB 3223 wascloned into the Xba I site such that the αICP4 promoter and thestructural sequences of the HBsAg gene were in the same transcriptionalorientation as determined from Eco RI DNA restriction endonucleasepatterns. Thus, by the foregoing procedure, chimeric gene Pα₄ -HBsAg wascloned into the Bgl II site of the TK gene.

The resulting recombinant plasmid pRB 3225 (ATCC Accession No. 39715)was cotransfected with intact HSV-1 (F) DNA in 143 tk- cells asdescribed above with reference to FIG. 1, and the resulting recombinantvirus R3225 (deposited on Jun. 4, 1984 ATCC Accession No. VR2087)containing a Pα ₄ -HBsAg fragment in the TK gene was selected from theTK- progeny in 143 tk- cells and checked for HBsAg expression as setforth below. The transfection and selection step is depictedschematically in FIG. 5.

To differentiate the expression of α-regulated HBsAg (R3225) from thatof β-regulated HBsAg (R3223) recombinant viruses, the observation that αgenes are transcribed in the absence of de-novo protein synthesispostinfection, whereas α genes require the presence of functional αgenes for their expression, was utilized according to the followingprocedure.

Replicate Hep-2 cell cultures in 25 cm² flasks were preincubated for 1hr. in maintenance medium containing 50 μg/ml of cycloheximide (+cyclo),and then infected at 20/pfu per cell with wild type [HSV-1(F)], R3222,R3223 or R3225 viruses, respectively. Five hours postinfection, themedium containing cycloheximide was removed and the cells wereextensively washed and then incubated in medium containing antinomycin D(10 μg/ml). The cells and medium were harvested after 90 minutes ofadditional incubation. Medium and cell lysate were assayed for HBsAg bythe AUSZYME II diagnostic kit (Abbott Laboratories). The controlexperiments (-cyclo) followed same washing procedure except that nodrugs were added. Results are given in Table 2, below.

                  TABLE 2                                                         ______________________________________                                        Regulation of HBsAg Expressed by Recombinant Viruses                          Conditions Materials                                                                              HSV-1                                                     of infection                                                                             tested   (F)     R3222 R3223 R3225                                 ______________________________________                                        -Cycloheximide                                                                           medium    <0.06* <0.06 5.22  2.34                                  -Cycloheximide                                                                           cell     <0.06   <0.06 3.09  1.04                                             lysate                                                             +Cycloheximide                                                                           medium   <0.06   <0.06 <0.06 0.77                                  +Cycloheximide                                                                           cell     <0.06   <0.06 <0.06 0.80                                             lysate                                                             ______________________________________                                         *Optical density units.                                                  

As shown above in Table 2, HBsAg was made by cells infected with R3225but not with R3223 after removal of inhibitory concentrations ofcycloheximide present in the medium during infection and for five hourspostinfection.

A characteristic of HSV-1 α genes is that they are the only viral genestranscribed in cells exposed during and after infection to inhibitors ofprotein synthesis. As shown in Table 2, above, only the HBsAg genecontained in R3225 was expressed in cells infected and maintained in thepresence of cycloheximide and then released from the inhibitory effectsof cycloheximide in the presence of actionomycin D to preclude thetranscription of β genes dependent on the synthesis of the αICP4 geneproduct.

Discussion of Results

The foregoing demonstrates that the HSC genome can act as an expressionvector for foreign genes and, in particular, for HBsAg.

Although HSV shuts off host macromolecular metabolism and especiallyhost protein synthesis, it does not adversely affect the expression offoreign genes (as exemplified by HBsAg) inserted into the viral genomeand regulated by HSV promoter-regulatory regions. The antigenicity ofthe gene product, its buoyant density in CsCl density gradients and thecharacteristic 18-25 nm particle size present in the banded preparationsdemonstrate that the product of the HBsAg gene carried by HSV is anauthentic product of that gene. The observation that HBsAg is excretedfrom the infected cell suggests that the antigen is processed properlyfor exportation from cells.

The results presented herein indicate that both the αICP4 and the βTKlinked HBsAg genes expressed the antigen for at least 12 hours. Thepatterns of synthesis of the HBsAg and the observation that αICP4 linkedwas regulated as an α gene indicate that the chimeric HBsAg genes in theHSV-1 vector were regulated as viral genes. The production of HBsAg canbe especially heightened by insertion of the gene under an αpromoter-regulatory region into the genome of ts mutants in the ICP4gene inasmuch as such mutants have been shown to express αgenescontinuously in cells infected and maintained at the non-permissivetemperature.

Use of the HSV genome as a vector for foreign genes is particularlyuseful for the biosynthesis in human cells and characterization ofproducts of (a) genes of viruses whose growth is restricted in cellculture (e.g., hepatitis B virus), (b) genes of infectious agents thatare particularly hazardous for humans, and (c) cellular genes expressedat very low levels, or not at all, in cultured cells. HSV-1 expressionvectors would also be useful for analyses of gene regulation, especiallyat the translational level.

A particular advantage of HSV-vectors relates to the fact that theseviruses have a very wide host range; infection of many different typesof cells in a uniform manner is made possible. Therefore, foreign genesinserted into an HSV-1 vector can be readily propagated serially inculture and will be packaged as part of the viral genome in virusparticles. The vector can then be used to infect synchronously largescale cell cultures to obtain sustained expression of the foreign genein all of the infected cells. This method has considerable advantageover other methods which rely upon transfection of cells with DNA andselection of a small minority of cells which express the foreign gene.This procedure is applicable for human diploid cell strains authorizedfor human vaccine production (e.g. MRC-5 or W138) which do not lendthemselves to transformation by DNA fragments for expression of foreigngenes.

In the exemplary illustration given herein, the HBsAg was inserted intowild type genomes modified at the site of insertion of the HBsAg gene.Although as much as 7 Kbp of DNA has been inserted previously (Knipe etal, Proc. Natl. Acad. Sci., Vol. 75, pp. 3896-3900, 1978), the capacityof wild type HSV DNA to carry additional gene products might be limited.

The construction of mutant HSV-1(F)I358 from which approximately 14 Kbpof DNA contained within the internal reiterated sequences had beenreplaced with a 2 Kbp insert has previously been reported (Poffenbergeret al, Proc. Natl. Acad. Sci., Vol. 80, pp. 2690-2694, 1983). Byreplacing the insert and expanding the genome to its known maximumcapacity, the I358 mutant could carry as much as 23 Kbp of foreign DNA.HSV-1(F)I358 has the capacity therefore to serve as a vector of severalgenes specifying antigens from a variety of human infectious agents forimmunoprophylaxis.

The DNA of Herpes Simplex virus, type 2, (HSV-2) is essentiallyidentical in structure to that of HSV-1, and differs only in nucleotidematching of base pairs. Therefore, DNA constructs identical to thoseillustrated herein using the HSV-1 genome are feasible according to thepresent invention.

Herpes Simplex virus 1 is readily available to the public. It is, forexample, on deposit with the American Type Culture Collection, 12301Parklawn Drive, Rockville, Md. 20852, USA, under ATCC Accession NumberVR733. Likewise, plasmid pBR322 is readily available to the public; ittoo is on deposit at the ATCC, under ATCC Accession Number 31344. TheATCC has been requested to maintain these cultures, inter alia, inaccordance with the requirements of the Implementing Regulations to theEuropean Patent Convention, as well as the remaining cultures identifiedherein by ATCC Accession Numbers, which have been deposited.

The remaining materials identified herein are publicly available. Forexample, the restriction enzymes, T4 DNA ligase, polynucleotide kinase,T4 DNA polymerase, exonuclease Bal 31, and restriction enzyme linkerswere purchased from New England Biolabs, Cambridge, Mass. USA and usedas directed by the manufacturer. DNA probes for screening E. colicolonies for desired recombinant plasmids were labeled by nicktranslation with [γ-P³² ]-ATP obtained from New England Nuclear,Cambridge, Mass. USA.

The F strain of HSV-1[HSV-1(F)] and all recombinants derived herein weregrown and titered on Vero or HEp-2 cell lines obtained from AmericanType Culture Collection. The rabbit skin cells used for transfectionwith viral DNA, as well as the human tk-cell line used for selection ofTK- recombinants, are publicly available.

The foregoing detailed description is given for clearness ofunderstanding only, and no unnecessary limitations should be inferredtherefrom, as modifications within the scope of the invention will beobvious to those skilled in the art.

I claim:
 1. A recombinant viral genome of a Herpes simplex virus (HSV)containing an expressable non-HSV nucleotide sequence encoding a desiredprotein permanently integrated at a non-lethal site of said genome. 2.The recombinant viral genome of claim 1 wherein said Herpes simplexvirus is HSV-1.
 3. The recombinant viral genome of claim 1 wherein saidnon-lethal site and said sequence are selected by the use of aselectable marker in said genome.
 4. The recombinant viral genome ofclaim 2 wherein said marker is the thymidine kinase (TK) gene of saidgenome.
 5. The recombinant viral genome of claim 3 wherein said sequenceis flanked at its respective ends with a suitable transcriptiontermination signal and a promoter-regulatory region comprising an HSValpha gene promoter.
 6. The recombinant viral genome of claim 5 whereinsaid promoter-regulatory region comprises an alpha gene promoter of aninfected cell protein gene.
 7. The recombinant viral genome of claim 6wherein said promoter-regulatory region comprises an alpha gene promoterof an infected cell protein number 4 gene.
 8. The recombinant viralgenome of claim 3 wherein said sequence is flanked at its respectiveends with a suitable transcription termination signal and apromoter-regulatory region comprising an HSV beta gene promoter.
 9. Therecombinant viral genome of claim 8 wherein said beta gene promoter is apromoter of the thymidine kinase gene of said genome.
 10. Therecombinant viral genome of claim 1 wherein said sequence comprises astructural coding sequence of hepatitis B surface antigen (HBsAg). 11.The recombinant viral genome of claim 1 wherein said Herpes simplexvirus is HSV-2.
 12. A plasmid vector containing an expressable non-HSVnucleotide sequence encoding a desired protein permanently integrated ata non-lethal site of a viral genome of a Herpes simplex virus (HSV). 13.The plasmid vector of claim 12 comprising plasmid pRB 3223 (ATCCAccession No. 39716).
 14. The plasmid vector of claim 12 comprisingplasmid pRB 3225 (ATCC Accession No. 39715).
 15. The plasmid vector ofclaim 12 wherein said Herpes simplex virus is HSV-1.
 16. The plasmidvector of claim 12 wherein said non-lethal site and said sequence areselected by the use of a selectable marker in said genome.
 17. Theplasmid vector of claim 16 wherein said marker is the thymidine kinase(TK) gene of said genome.
 18. The plasmid vector of claim 16 whereinsaid sequence is flanked at its respective ends with a suitabletranscription termination signal and a promoter-regulatory regioncomprising an HSV alpha gene promoter.
 19. The plasmid vector of claim18 wherein said promoter-regulatory region comprises an alpha genepromoter of an infected cell protein.
 20. The plasmid vector of claim 19wherein said promoter-regulatory region comprises an alpha gene promoterof an infected cell protein number 4 gene.
 21. The plasmid vector ofclaim 16 wherein said sequence is flanked at its respective ends with asuitable transcription termination signal and a promoter-regulatoryregion comprising an HSV beta gene promoter.
 22. The plasmid vector ofclaim 21 wherein said beta gene promoter is a promoter of the thymidinekinase gene of said genome.
 23. The plasmid vector of claim 12 whereinsaid sequence comprises a structural coding sequence for hepatitis Bsurface antigen (HBsAg).
 24. The plasmid vector of claim 12 wherein saidHerpes simplex virus is HSV-2.
 25. A recombinant virus comprising aproduct of cotransfection of the plasmid vector of claim 12 with intactHerpes simplex virus DNA in a suitable host and screening of resultingprogeny for progeny containing said sequence.
 26. The recombinant virusof claim 25 comprising recombinant virus R3223 (ATCC Accession No.VR2086).
 27. The recombinant virus of claim 25 comprising recombinantvirus R3225 (ATCC Accession No. VR2087).
 28. The recombinant virus ofclaim 25 wherein said non-lethal site and said sequence are selected bythe use of a selectable marker in said genome, and said progeny arescreened by selection for the presence or absence of said marker.
 29. Amethod of obtaining a desired protein, comprising the steps of:(a)preparing a plasmid vector according to claim 12 wherein saidexpressible non-HSV nucleotide sequence codes for the expression of saidprotein; (b) cotransfecting said plasmid vector with intact Herpessimplex virus DNA to produce viral progeny; (c) screening and isolatingprogeny containing said sequence to obtain a recombinant virus; and, (d)infecting a suitable host with said recombinant virus and culturing saidinfected cells whereby said protein is produced.
 30. The method of claim29 including the further steps of harvesting and purifying said protein.31. The method of claim 29 wherein said Herpes Simplex virus of saidvector is HSV-1.
 32. The method of claim 29 wherein said non-lethal siteand said sequence are selected by the use of a selectable marker in saidgenome, and said screening step (c) is carried out by selecting for thepresence or absence of said marker.
 33. The method of claim 32 whereinsaid marker is the thymidine kinase (TK) gene of said genome.
 34. Themethod of claim 32 wherein said sequence is flanked at its respectiveends with a suitable transcription termination signal and apromoter-regulatory region comprising an HSV alpha gene promoter. 35.The method of claim 34 wherein said promoter-regulatory region comprisesan alpha gene promoter of an infected cell protein gene.
 36. The methodof claim 35 wherein said promoter-regulatory region comprises an alphagene promoter of an infected cell protein number 4 gene.
 37. The methodof claim 32 wherein said sequence is flanked at its respective ends witha suitable transcription termination signal and a promoter-regulatoryregion comprising an HSV beta gene promoter.
 38. The method of claim 37wherein said beta gene promoter is a promoter of the thymidine kinasegene of said genome.
 39. The method of claim 29 wherein said sequencecomprises a structural coding sequence for hepatitis B surface antigen(HBsAg).
 40. The method of claim 29 wherein said Herpes Simplex virus ofsaid vector is HSV-2.
 41. A method of preparing a recombinant viralgenome of a Herpes Simplex virus (HSV), said method comprising the stepsof:(a) isolating a viral genome of said Herpes Simplex virus; and, (b)permanently integrating into said genome at a non-lethal site thereof anexpressable non-HSV nucleotide sequence encoding a desired protein. 42.The method of claim 41 wherein said Herpes Simplex virus is HSV-1. 43.The method of claim 41 wherein said non-lethal site and said sequenceare selected by the use of a selectable marker in said genome.
 44. Themethod of claim 43 wherein said marker is the thymidine kinase (TK) geneof said genome.
 45. The method of claim 43 wherein said sequence isflanked at its respective ends with a suitable transcription terminationsignal and a promoter-regulatory region comprising an HSV alpha genepromoter.
 46. The method of claim 45 wherein said promoter-regulatoryregion comprises an alpha gene promoter of an infected cell protein. 47.The method of claim 46 wherein said promoter-regulatory region comprisesan alpha promoter of an infected cell protein number 4 gene.
 48. Themethod of claim 41 wherein said sequence is flanked at its respectiveends with a suitable transcription termination signal and apromoter-regulatory region comprising an HSV beta gene promoter.
 49. Themethod of claim 48 wherein said beta gene promoter is a promoter of thethymidine kinase gene of said genome.
 50. The method of claim 41 whereinsaid sequence comprises a structural coding sequence for hepatitis Bsurface antigen (HBsAg).
 51. The method of claim 41 wherein said HerpesSimplex virus is HSV-2.
 52. A method of obtaining a recombinant virus,comprising the steps of :(a) cotransfecting the plasmid vector of claim12 with intact Herpes Simplex virus DNA in a suitable host; and, (b)screening the resulting progeny for a recombinant virus containing saidexpressible non-HSV nucleotide sequence.
 53. The method of claim 52wherein said Herpes Simplex virus is HSV-1.
 54. The method of claim 52wherein said non-lethal site and said sequence are selected by the useof a selectable marker in said genome, and said screening step (b) iscarried out by selecting for the presence or absence of said marker. 55.The method of claim 54 wheren said marker is the thymidine kinase (TK)gene of said genome.
 56. The method of claim 54 wherein said sequence isflanked at its respective ends with a suitable transcription terminationsignal and a promoter-regulatory region comprising an HSV alpha genepromoter.
 57. The method of claim 56 wherein said promoter-regulatoryregion comprises an alpha gene promoter of an infected cell protein. 58.The method of claim 57 wherein said promoter-regulatory region comprisesan alpha gene promoter of an infected cell protein number 4 gene. 59.The method of claim 54 wherein said sequence is flanked at itsrespective ends with a suitable transcription termination signal and apromoter-regulatory region comprising an HSV beta gene promoter.
 60. Themethod of claim 59 wherein said beta gene promoter is a promoter of thethymidine kinase gene of said genome.
 61. The method of claim 52 whereinsaid sequence comprises a structural coding sequence for hepatitis Bsurface antigen (HBsAg).
 62. The method of claim 52 wherein said HerpesSimplex virus of said vector is HSV-2.